Reversal of viral-induced systemic shock and respiratory distress by blockade of the lymphotoxin beta pathway

ABSTRACT

This invention provides methods of inducing an antiviral response in an individual comprising administering to the individual an effective amount of a LT-B blocking agent and a pharmaceutically acceptable carrier. In particular this invention provides methods for treating viral-induced systemic shock and respiratory distress.

RELATED APPLICATIONS

[0001] This is a continuation of PCT/US99/23477, filed on Oct. 8, 1999as a continuation-in-part of prior U.S. Provisional Ser. No. 60/103,662filed Oct. 9, 1998. The teachings of the earlier-filed Provisionalpatent application are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates generally to methods of inducing anantiviral response in an individual. In particular, this inventionprovides methods for treating viral-induced systemic shock andrespiratory distress in an individual. The methods involvesadministration of certain “lymphotoxin-beta blocking agents”.

BACKGROUND OF THE INVENTION

[0003] Several viruses including Sin Nombre (SNV), Ebola, Marburg,Lassa, and Dengue all cause acute diseases with many of the followingsymptoms: rapid onset, fever, systemic shock, and pulmonary distress(Lacy et al. (1997) Adv. Ped. Inf. Dis. 12:21). Another commonalityamong these infections is the systemic distribution of viral infection,targeting endothelial cells and macrophages (Lacy et al. (1997) Adv.Ped. Inf. Dis. 12:21). Most of these emerging viruses, with theexception of SNV, were initially identified decades ago. In the yearssince their discovery these pathogens have re-emerged in outbreaksworldwide. As of June 1998 there have been 183 confirmed cases of SNV,the causative agent of Hantavirus Pulmonary Shock Syndrome, in thesouthwestern United States due to an increase in deer mouse populations.Only 55% of these cases have survived infection (Centers for DiseaseControl and Prevention. MMWR. 47, 449 (1998)). Little is currently knownabout the pathogenesis of these viruses nor how to effectively treat thethousands of patients infected globally each year suffering fromviral-induced systemic shock and respiratory distress.

[0004] Thus, there exists a need to identify novel methods for treatingviral-induced systemic shock and respiratory distress in an individual.

SUMMARY OF THE INVENTION

[0005] The present invention solves the problem referred to above byproviding pharmaceutical compositions and methods for treatingviral-induced systemic shock and respiratory distress in an individual.

[0006] The methods and compositions of this invention capitalize in parton the discovery that certain agents, defined herein as lymphotoxin-beta(LT-B) blocking agents may be used in treating viral-induced systemicshock and respiratory distress in an individual. In a one embodiment,the LT-B blocking agents is a lymphotoxin-beta receptor (LT-B-R)blocking agent. In a preferred embodiment, the LT-B-R is an antibodyagainst a lymphotoxin-B receptor or a soluble lymphotoxin B receptor. Ina most preferred embodiment, the LT-B-R blocking agent is a recombinantLT-B-R fusion protein that has an LT-B-R extracellular ligand bindingdomain fused to an immunoglobulin constant heavy chain domain.

[0007] The foregoing and other objects, features, aspects and advantagesof the present invention, as well as the invention itself, will be morefully understood from the following description of preferredembodiments.

BRIEF DESCRIPTION OF THE FIGURES

[0008]FIG. 1 shows that infection of NZB mice with Clone 13 LCMV resultsin mortality. Mortality curve of NZB mice infected with LCMV-13 (n=14)and viral titers in various tissues of LCMV-13 (n=7) infected mice sixdays post-infection.

[0009]FIG. 2 shows the histological profile of LCMV-13 infection in NZBmice. (A) Normal lung at (100×, H+E) (B) Interstitial pneumonitis withmononuclear cell infiltrate and alveolar wall thickening in the lung,day 5 post-infection (100×, H+E) (C) Lymphoid depletion, cellularnecrosis and obliteration of follicular architecture in the spleen (25×,H+E) (D) Higher magnification showing cellular necrosis andkaryorrhectic debris in the spleen (158, H+E) (E) LCMV-13 positiveendothelial cells (arrows) and macrophages (white arrows) in the lung(100×, IHC) (F) LCMV-13 positive endothelial cells endothelial cells(arrows) and mesothelial cells (arrow heads), and macrophages (whitearrows) in the spleen (50×, IHC) (G) LCMV-13 positive endothelial cellsin the heart (100×, IHC) (H) LCMV-13 positive Kupffer cells andsinusoidal lining cells in the liver (100×, IHC).

[0010]FIG. 3 shows that blockage of the LTβPR signaling pathwayssignificantly improves survival rates among Clone 13 infected NZB mice.Mortality curves for Clone 13 infected NZB mice treated as described arepresented here. NZB mice were given 2.5×10⁶ pfu Cl 13 i.v. followed bytwo i.p. injections containing 250 μg of TN3-19.12 antibody in endotoxinfree PBS (see reference S) on days 1 and day 4 post-infection. Controlmice were injected with the same volume of PBS lacking antibody on thesame days. Mice were treated as described in reference R. For the tripletreated group, TNFR55-Ig and LTβR-Ig proteins were given on day 0 andday 3 post-infection, i.p., in 200 μg amounts. Control mice were givenhuman antibody used in the synthesis of these fusion proteins(AY1943-29) on the same days in identical amounts. Mice receivingLTβR-Ig only were treated identically, except the TNFR55-Ig injectionswere omitted. Data was compiled from several experiments anti-TNF(TN3-19.12) alone, n=16 for LTβR-Ig alone, n=10 for the triple treatmentgroup, (n=10 for the triple treatment group, n=22 for LTβR-Ig alone,n=10 for the LTβR-Ig+TNFR55-Ig group, n=5 for the anti-TNF and TNFR55-Igtreated group, n=6 for anti-TNF (TN3-19.12) alone, and n=25 forControl).

[0011]FIG. 4 shows that blockage of the LTβR pathway results in adecrease in CD8 T cell function. Splenocytes from mice in differenttreatment groups were harvested on day 6 post-infection and stained withan L^(d) tetramer containing a NP118 9mer peptide as previouslydescribed. Values given are adjusted for non-specific backgroundstaining. To monitor interferon gamma production in response to the samepeptide, cells were incubated for 5 hours at 37° C. in the presence ofNP118 at 0.1 μg/ml final concentration and IL-2. Values given here areadjusted for background levels in the absence of peptide. Spleenocytesfrom three mice treated with control human Ig were pooled as were thosefrom two LTβR-Ig mice (LT beta #2/3). All other results are fromindividual mice.

[0012]FIG. 5 shows that depletion of CD8⁺T cells, not CD4⁺T cells,reverses the lethal effects of LCMV-13 infection in NZB mice. Mice weretreated as described for depletion of cell populations in vivo. Amortality curve is presented for each of the treated groups (n=4).

DETAILED DESCRIPTION OF THE INVENTION

[0013] Definitions

[0014] In order to more clearly and concisely point out the subjectmatter of the claimed invention, the following definitions are providedfor specific terms used in the following written description andappended claims.

[0015] Lymphotoxin-beta (LT-beta) is a member of the TNF family ofligands, which also includes the ligands to the Fas, CD27, CD30, CD40,OX-40 and 4-1BB receptors (Smith et al., Cell, 76, pp. 959-62 (1994)).Signaling by several members of the TNF family-including TNF, LT-alpha,LT-beta and Fas-can induce tumor cell death by necrosis or apoptosis(programmed cell death). In non-tumorigenic cells, TNF and many of theTNF family ligand-receptor interactions influence immune systemdevelopment and responses to various immune challenges.

[0016] Lymphotoxin-beta (also called p33), has been identified on thesurface of T lymphocytes, T cell lines, B cell lines andlymphokine-activated killer (LAK) cells. LT-beta is the subject ofapplicants' co-pending international applications PCT/US91/04588,published Jan. 9, 1992 as WO 92/00329; and PCT/US93/11669, publishedJun. 23, 1994 as WO 94/13808, which are herein incorporated byreference.

[0017] The LT-beta receptor, a member of the TNF family of receptors,specifically binds to surface LT ligands. LT-beta-R binds LT heteromericcomplexes (predominantly LT-alpha 1/beta 2 and LT-alpha 2/beta 1) butdoes not bind TNF or LT-alpha (Crowe et al., Science, 264, pp. 707-10(1994)). Signaling by LT-beta-R may play a role in peripheral lymphoidorgan development and in humoral immune responses.

[0018] LT-beta-R mRNAs are found in human spleen, thymus and other majororgans. LT-beta-R expression patterns are similar to those reported forp55-TNF-R except that LT-beta-R is lacking on peripheral blood T cellsand T cell lines.

[0019] The term “LT-beta-blocking agent” refers to an agent that candiminish ligand binding to LT-beta, cell surface LT-beta clustering orLT-beta signalling, or that can influence how the LT-beta signal isinterpreted within the cell. Examples of LT-beta blocking agents includeanti-LT-beta, soluble LT-beta-R-Fc molecules, and anti-LT-alpha,anti-LT-alpha/beta and anti-LT-beta-R Abs. Preferably, the antibodies donot cross-react with the secreted form of LT-alpha.

[0020] The term “LT-beta-receptor blocking agent” refers to an agentthat can diminish ligand binding to LT-beta-R, cell surface LT-beta-Rclustering or LT-beta-R signalling, or that can influence how theLT-beta-R signal is interpreted within the cell. Examples of LT-beta-Rblocking agents include soluble LT-beta-R-Fc molecules, and andanti-LT-beta-R Abs. Preferably, the antibodies do not cross-react withthe secreted form of LT-alpha.

[0021] The term “anti-LT-beta receptor antibody” refers to any antibodythat specifically binds to at least one epitope of the LT-beta receptor.

[0022] The term “anti-LT antibody” refers to any antibody thatspecifically binds to at least one epitope of LT-alpha , LT-beta or aLT-alpha/beta complex.

[0023] The term “LT ligand” refers to a LT heteromeric complex orderivative thereof that can specifically bind to the LT-beta receptor.

[0024] The term “LT-beta-R signaling” refers to molecular reactionsassociated with the LT-beta-R pathway and subsequent molecular reactionswhich result therefrom.

[0025] The term “LT-beta-R ligand binding domain” refers to the portionor portions of the LT-beta-R that are involved in specific recognitionof and interaction with a LT ligand.

[0026] The terms “LT-alpha/beta heteromeric complex” and “LT heteromericcomplex” refer to a stable association between at least one LT-alpha andone or more LT-beta subunits, including soluble, mutant, altered andchimeric forms of one or more of the subunits. The subunits canassociate through electrostatic, van der Waals, or covalentinteractions. Preferably, the LT-alpha/62 heteromeric complex has atleast two adjacent LT-beta subunits and lacks adjacent LT-alphasubunits. When the LT-alpha/beta heteromeric complex serves as aLT-beta-R activating agent in a cell growth assay, the complex ispreferably soluble and has the stoichiometry LT-alpha 1/beta 2.

[0027] Soluble LT-alpha/62 heteromeric complexes lack a transmembranedomain and can be secreted by an appropriate host cell which has beenengineered to express LT-alpha and/or LT-beta subunits (Crowe et al., J.Immunol. Methods, 168, pp. 79-89 (1994)).

[0028] The terms “surface LT-alpha/62 complex” and “surface LT complex”refer to a complex comprising LT-alpha and membrane-bound LT-betasubunits—including mutant, altered and chimeric forms of one or more ofthe subunits—which is displayed on the cell surface. “Surface LT ligand”refers to a surface LT complex or derivative thereof that canspecifically bind to the LT-beta receptor.

[0029] An “effective amount” is an amount sufficient to effectbeneficial or desired clinical results. An effective amount can beadministered in one or more administrations. For purposes of thisinvention, an effective amount of an agent which blocks the binding oflymphotoxin-B to its receptor is an amount of the agent that issufficient to ameliorate, stabilize, or delay the development of a viralresponse. In particular, an agent that is sufficient to ameliorate,stabilize, or delay the development of viral-induced systemic shock andrespiratory distress. Detection and measurement of these indicators ofefficacy are known to those of skill in the art.

[0030] An “individual” refers to vertebrates, particularly members of amammalian species, and includes but is not limited to domestic animals,sports animals, and primates, including humans.

[0031] “functional equivalent” of an amino acid residue is (i) an aminoacid having similar reactive properties as the amino acid residue thatwas replaced by the functional equivalent; (ii) an amino acid of anantagonist of the invention, the amino acid having similar properties asthe amino acid residue that was replaced by the functional equivalent;(iii) a non-amino acid molecule having similar properties as the aminoacid residue that was replaced by the functional equivalent.

[0032] A first polynucleotide encoding a proteinaceous antagonist of theinvention is “functionally equivalent” compared with a secondpolynucleotide encoding the antagonist protein if it satisfies at leastone of the following conditions:

[0033] (a): the “functional equivalent” is a first polynucleotide thathybridizes to the second polynucleotide under standard hybridizationconditions and/or is degenerate to the first polynucleotide sequence.Most preferably, it encodes a mutant protein having the activity of anintegrin antagonist protein;

[0034] (b) the “functional equivalent” is a first polynucleotide thatcodes on expression for an amino acid sequence encoded by the secondpolynucleotide.

[0035] “functional equivalent” of an amino acid residue is (i) an aminoacid having similar reactive properties as the amino acid residue thatwas replaced by the functional equivalent; (ii) an amino acid of anantagonist of the invention, the amino acid having similar properties asthe amino acid residue that was replaced by the functional equivalent;(iii) a non-amino acid molecule having similar properties as the aminoacid residue that was replaced by the functional equivalent.

[0036] A first polynucleotide encoding a proteinaceous antagonist of theinvention is “functionally equivalent” compared with a secondpolynucleotide encoding the antagonist protein if it satisfies at leastone of the following conditions:

[0037] (a): the “functional equivalent” is a first polynucleotide thathybridizes to the second polynucleotide under standard hybridizationconditions and/or is degenerate to the first polynucleotide sequence.Most preferably, it encodes a mutant protein having the activity of anintegrin antagonist protein;

[0038] (b) the “functional equivalent” is a first polynucleotide thatcodes on expression for an amino acid sequence encoded by the secondpolynucleotide.

[0039] The LT-B blocking agents used in the invention include, but arenot limited to, the agents listed herein as well as their functionalequivalents. As used herein, the term “functional equivalent” thereforerefers to a LT-B blocking agent or a polynucleotide encoding the LT-Bblocking agent that has the same or an improved beneficial effect on therecipient as the LT-B blocking agent of which it is deemed a functionalequivalent. As will be appreciated by one of ordinary skill in the art,a functionally equivalent protein can be produced by recombinanttechniques, e.g., by expressing a “functionally equivalent DNA”.Accordingly, the instant invention embraces LT-B blocking agent encodedby naturally-occurring DNAs, as well as by non-naturally-occurring DNAswhich encode the same protein as encoded by the naturally-occurring DNA.Due to the degeneracy of the nucleotide coding sequences, otherpolynucleotides may be used to encode LT-B blocking agents. Theseinclude all, or portions of the above sequences which are altered by thesubstitution of different codons that encode the same amino acid residuewithin the sequence, thus producing a silent change. Such alteredsequences are regarded as equivalents of these sequences. For example,Phe (F) is coded for by two codons, TTC or TTT, Tyr (Y) is coded for byTAC or TAT and His (H) is coded for by CAC or CAT. On the other hand,Trp (W) is coded for by a single codon, TGG. Accordingly, it will beappreciated that for a given DNA sequence encoding a particular integrinthere will be many DNA degenerate sequences that will code for it. Thesedegenerate DNA sequences are considered within the scope of thisinvention.

[0040] The term “fusion” or “fusion protein” refers to a co-linear,covalent linkage of two or more proteins or fragments thereof via theirindividual peptide backbones, most preferably through genetic expressionof a polynucleotide molecule encoding those proteins. It is preferredthat the proteins or fragments thereof are from different sources sothat this type of fusion protein is called a “chimeric” molecule. Thus,preferred fusion proteins are chimeric proteins that include a LT-Bblocking agent or fragment covalently linked to a second moiety that isnot a LT-B blocking agent. Preferred fusion proteins of the inventionmay include portions of intact antibodies that retain antigen-bindingspecificity, for example, Fab fragments, Fab′ fragments, F(ab′)2fragments, F(v) fragments, heavy chain monomers or dimers, light chainmonomers or dimers, dimers consisting of one heavy and one light chain,and the like.

[0041] The most preferred fusion proteins are chimeric and comprise aLT-B blocking agent moiety fused or otherwise linked to all or part ofthe hinge and constant regions of an immunoglobulin light chain, heavychain, or both. Thus, this invention features a molecule which includes:(1) a LT-B blocking agent moiety, (2) a second peptide, e.g., one whichincreases solubility or in vivo life time of the LT-B blocking agentmoiety, e.g., a member of the immunoglobulin super family or fragment orportion thereof, e.g., a portion or a fragment of IgG, e.g., the humanIgG1 heavy chain constant region, e.g., CH2, CH3, and hinge regions.Specifically, a “LT-B or LT-B-R/Ig fusion” is a protein comprising abiologically active LT-B blocking of the invention (e.g. a solubleLT-B-R, or a biologically active fragment thereof linked to anN-terminus of an immunoglobulin chain wherein a portion of theN-terminus of the immunoglobulin is replaced with the LT-B blockingagent. A species of LT-B or LT-B-R/Ig fusion is an “LT-B-R/Fc fusion”which is a protein comprising an LT-B-R of the invention linked to atleast a part of the constant domain of an immunoglobulin. A preferred Fcfusion comprises a LT-B blocking agent of the invention linked to afragment of an antibody containing the C terminal domain of the heavyimmunoglobulin chains.

[0042] “standard hybridization conditions”—salt and temperatureconditions substantially equivalent to 0.5×SSC to about 5×SSC and 65° C.for both hybridization and wash. The term “standard hybridizationconditions” as used herein is therefore an operational definition andencompasses a range of hybridization conditions. Higher stringencyconditions may, for example, include hybridizing with plaque screenbuffer (0.2% polyvinylpyrrolidone, 0.2% Ficoll 400; 0.2% bovine serumalbumin, 50 mM Tris-HCl (pH 7.5); 1 M NaCl; 0.1% sodium pyrophosphate;1% SDS); 10% dextran sulfate, and 100 μg/ml denatured, sonicated salmonsperm DNA at 65° C. for 12-20 hours, and washing with 75 mM NaCl/7.5 mMsodium citrate (0.5×SSC)/1% SDS at 65° C. Lower stringency conditionsmay, for example, include hybridizing with plaque screen buffer, 10%dextran sulfate and 110 μg/ml denatured, sonicated salmon sperm DNA at55° C. for 12-20 hours, and washing with 300 mM NaCl/30 mM sodiumcitrate (2.0×SSC)/1% SDS at 55° C. See also Current Protocols inMolecular Biology, John Wiley & Sons, Inc. New York, Sections6.3.1-6.3.6, (1989).

[0043] A “therapeutic composition” as used herein is defined ascomprising the proteins of the invention and other biologicallycompatible ingredients. The therapeutic composition may containexcipients such as water, minerals and carriers such as protein.

[0044] II. Description of the Preferred Embodiments

[0045] The present invention depends in part upon the discovery thatLT-B blocking agents can induce an antiviral response in an individual.It was found that treating an individual infected with a virus cangreatly increase the survival rate of the individual. Specifically, itwas shown that treating LCMV-13 infected NZB mice with a LT-B blockingagent, such as LTβR-Ig fusion protein increased their survival rate 73%.

[0046] Currently treatment for Ebola, Dengue, SNV and other virusesmentioned herein is preventative via education on transmission ofdisease. Vaccines do not exist for these highly pathogenic viruses.Ribavirin, a guanidine analog, has been employed as a generic antiviraldrug to several of these infections with reproducible success onlydocumented in treatment of Lassa Fever when used early on in the illness(M. D. Lacy and R. A. Smego, Adv. Ped. Inf. Dis., 12, 21 (1997). Ourdata indicate that some of the pathology associated with these virusesmay be immune mediated. Blockade of the LT system could greatly increasethe chance for survival by transiently reducing virus specific CD8 Tcells numbers and their functionality. Clinical trials that employseveral means of blocking the TNFα pathway are already underway for thetreatment of several ailments (H. I. Pass, D. Mew, H. A. Pass, et al.,Chest Surg. Clin. N. Amer. 5, 73 (1995). We believe the LTβR-Igtreatment should be considered for further testing in animal models foreventual use in human trials involving patients with acute, rapidlyprogressive viral infections involving shock and/or pulmonary distress.

[0047] LT-B Blocking Agents

[0048] In one embodiment of this invention, the LT-beta blocking agentcomprises an antibody (Ab) directed against LT-beta that inhibitsLT-beta signaling. Preferably, the anti-LT-beta Ab is a monoclonalantibody (mAb). Inhibitory anti-LT-beta Abs and other LT-beta blockingagents can be identified using screening methods that detect the abilityof one or more agents to bind to a LT ligand, or to inhibit the effectsof LT-beta signalling on cells.

[0049] In another embodiment of this invention, the LT-beta blockingagent comprises an LT-beta receptor (LT-B-R) blocking agent. In apreferred embodiment, the LT-B-R blocking agent is an antibody (Ab)directed against LT-beta-R that inhibits LT-beta-R signaling.Preferably, the anti-LT-beta-R Ab is a monoclonal antibody (mAb). Onesuch inhibitory anti-LT-beta-R mAb is BDA8 mAb. Inhibitoryanti-LT-beta-R Abs and other LT-beta-R blocking agents can be identifiedusing screening methods that detect the ability of one or more agentseither to bind to the LT-beta-R or LT ligand, or to inhibit the effectsof LT-beta-R signalling on cells.

[0050] One screening method makes use of the cytotoxic effects ofLT-beta-R signalling on tumor cells bearing the LT-beta-R. Tumor cellsare exposed to one or more LT-beta-R activating agents to induceLT-beta-R signalling. LT-beta-R activating agents include LT-alpha/62heteromeric complexes (preferably soluble LT-alpha 1/beta 2) in thepresence of IFN-gamma, or an activating anti-LT-beta-R Ab (see below;also described in applicants' co-pending U.S. application Ser. No.08/378,968).

[0051] Antibodies and other agents that can block LT-beta-R signallingare selected based on their ability to inhibit the cytotoxic effect ofLT-beta-R signalling on tumor cells in the following assay:

[0052] 1) Tumor cells such as HT29 cells are cultured for three to fourdays in a series of tissue culture wells containing media and at leastone LT-beta-R activating agent in the presence or absence of serialdilutions of the agent being tested;

[0053] 2) A vital dye stain which measures mitochondrial function suchas MTT is added to the tumor cell mixture and reacted for several hours;

[0054] 3) The optical density of the mixture in each well is quantitatedat 550 nm wavelength light (OD 550). The OD 550 is proportional to thenumber of tumor cells remaining in the presence of the LT-beta-Ractivating agent and the test LT-beta-R blocking agent in each well. Anagent or combination of agents that can reduce LT-beta-R-activated tumorcell cytotoxicity by at least 20% in this assay is a LT-beta-R blockingagent within the scope of this invention.

[0055] Any agent or combination of agents that activate LT-beta-Rsignalling can be used in the above assay to identify LT-beta-R blockingagents. LT-beta-R activating agents that induce LT-beta-R signalling(such as activating anti-LT-beta-R mAbs) can be selected based on theirability—alone or in combination with other agents—to potentiate tumorcell cytotoxicity using the tumor cell assay described above.

[0056] Another method for selecting an LT-beta-R blocking agent is tomonitor the ability of the putative agent to directly interfere with LTligand-receptor binding. An agent or combination of agents that canblock ligand-receptor binding by at least 20% is an LT-beta-R blockingagent within the scope of this invention.

[0057] Any of a number of assays that measure the strength ofligand-receptor binding can be used to perform competition assays withputative LT-beta-R blocking agents. The strength of the binding betweena receptor and ligand can be measured using an enzyme-linkedimmunoadsorption assay (ELISA) or a radio-immunoassay (RIA). Specificbinding may also be measured by fluorescently labelling antibody-antigencomplexes and performing fluorescence-activated cell sorting (FACS)analysis, or by performing other such immunodetection methods, all ofwhich are techniques well known in the art.

[0058] The ligand-receptor binding interaction may also be measured withthe BIAcore TM instrument (Pharmacia Biosensor) which exploits plasmonresonance detection (Zhou et al., Biochemistry, 32, pp. 8193-98 (1993);Faegerstram and O'Shannessy, “Surface plasmon resonance detection inaffinity technologies”, in Handbook of Affinity Chromatography, pp.229-52, Marcel Dekker, Inc., New York (1993)).

[0059] The BIAcore TM technology allows one to bind receptor to a goldsurface and to flow ligand over it. Plasmon resonance detection givesdirect quantitation of the amount of mass bound to the surface in realtime. This technique yields both on and off rate constants and thus aligand-receptor dissociation constant and affinity constant can bedirectly determined in the presence and absence of the putativeLT-beta-R blocking agent.

[0060] With any of these or other techniques for measuringreceptor-ligand interactions, one can evaluate the ability of aLT-beta-R blocking agent, alone or in combination with other agents, toinhibit binding of surface or soluble LT ligands to surface or solubleLT-beta-R molecules. Such assays may also be used to test LT-beta-Rblocking agents or derivatives of such agents (e.g. fusions, chimeras,mutants, and chemically altered forms)-alone or in combination-tooptimize the ability of that altered agent to block LT-beta-Ractivation.

[0061] The LT-beta-R blocking agents in one embodiment of this inventioncomprise soluble LT-beta receptor molecules. The sequence of theextracellular portion of the human LT-beta-R, which encodes the ligandbinding domain is shown in FIG. 1 of U.S. Pat. No. 5,925,351,incorporated by reference herein. Using the sequence information in FIG.1 of U.S. Pat. No. 5,925,351 and recombinant DNA techniques well knownin the art, functional fragments encoding the LT-beta-R ligand bindingdomain can be cloned into a vector and expressed in an appropriate hostto produce a soluble LT-beta-R molecule. Soluble LT-beta-R moleculesthat can compete with native LT-beta receptors for LT ligand bindingaccording to the assays described herein are selected as LT-beta-Rblocking agents.

[0062] A soluble LT-beta receptor comprising amino acid sequencesselected from those shown in FIG. 1 of U.S. Pat. No. 5,925,351 may beattached to one or more heterologous protein domains (“fusion domain”)to increase the in vivo stability of the receptor fusion protein, or tomodulate its biological activity or localization. Preferably, stableplasma proteins-which typically have a half-life greater than 20 hoursin the circulation-are used to construct the receptor fusion proteins.Such plasma proteins include but are not limited to: immunoglobulins,serum albumin, lipoproteins, apolipoproteins and transferrin. Sequencesthat can target the soluble LT-beta-R molecule to a particular cell ortissue type may also be attached to the LT-beta-R ligand binding domainto create a specifically-localized soluble LT-beta-R fusion protein. Allor a functional portion of the LT-beta-R extracellular region (FIG. 1 ofUS Pat. No 5,925,351) comprising the LT-beta-R ligand binding domain maybe fused to an immunoglobulin constant region like the Fc domain of ahuman IgG1 heavy chain (Browning et al., J. Immunol., 154, pp. 33-46(1995)). Soluble receptor-IgG fusion proteins are common immunologicalreagents and methods for their construction are known in the art (seee.g., U.S. Pat. No. 5,225,538). A functional LT-beta-R ligand bindingdomain may be fused to an immunoglobulin (Ig) Fc domain derived from animmunoglobulin class or subclass other than IgG1. The Fc domains ofantibodies belonging to different Ig classes or subclasses can activatediverse secondary effector functions. Activation occurs when the Fcdomain is bound by a cognate Fc receptor. Secondary effector functionsinclude the ability to activate the complement system, to cross theplacenta, and to bind various microbial proteins. The properties of thedifferent classes and subclasses of immunoglobulins are described inRoitt et al., Immunology, p. 4.8 (Mosby-Year Book Europe Ltd., 3d ed.1993). The complement enzyme cascade can be activated by the Fc domainsof antigen-bound IgG1 , IgG3 and IgM antibodies. The Fc domain of IgG2appears to be less effective, and the Fc domains of IgG4, IgA, IgD andIgE are ineffective at activating complement. Thus one can select a Fcdomain based on whether its associated secondary effector functions aredesirable for the particular immune response or disease being treatedwith the LT-beta-R-Fc fusion protein. If it would be advantageous toharm or kill the LT ligand-bearing target cell, one could select anespecially active Fc domain (IgG1) to make the LT-beta-R-Fc fusionprotein. Alternatively, if it would be desirable to targettheLT-beta-R-Fc fusion to a cell without triggering the complementsystem, an inactive IgG4 Fc domain could be selected.

[0063] Mutations in Fc domains that reduce or eliminate binding to Fcreceptors and complement activation have been described (S. Morrison,Annu. Rev. Immunol., 10, pp. 239-65 (1992)). These or other mutationscan be used, alone or in combination, to optimize the activity of the Fcdomain used to construct the LT-beta-R-Fc fusion protein.

[0064] The production of a soluble human LT-beta-R fusion proteincomprising ligand binding sequences fused to a human immunoglobulin Fcdomain (hLT-beta-R-Fc) is described in Example 1 of U.S. Pat. No.5,925,351 incorporated by reference herein. One CHO line made accordingto Example 1 that secretes hLT-beta-R-Fc is called “hLT beta ;R-hG1CHO#14”. A sample of this line was deposited on Jul. 21, 1995 with theAmerican Type Culture Collection (ATCC) (Rockville, Md.) according tothe provisions of the Budapest Treaty and was assigned the ATCCaccession number CRL 11965.

[0065] The production of a soluble murine LT-beta-R fusion molecule(mLT-beta-R-Fc) is described in Example 2 of U.S. Pat. No. 5,925,351incorporated by reference herein. A CHO line made according to Example 2of U.S. Pat. No. 5,925,351 that secretes mLT-beta-R-Fc is called “mLTbeta ;R-hG1 CHO#1.3.BB ”. A sample of this line was deposited on Jul.21, 1995 with the American Type Culture Collection (ATCC) (Rockville,Md.) according to the provisions of the Budapest Treaty and was assignedthe ATCC accession number CRL 11964.

[0066] Different amino acid residues forming the junction point of thereceptor-Ig fusion protein may alter the structure, stability andultimate biological activity of the soluble LT-beta receptor fusionprotein. One or more amino acids may be added to the C-terminus of theselected LT-beta-R fragment to modify the junction point with theselected fusion domain.

[0067] The N-terminus of the LT-beta-R fusion protein may also be variedby changing the position at which the selected LT-beta-R DNA fragment iscleaved at its 5′ end for insertion into the recombinant expressionvector. The stability and activity of each LT-beta-R fusion protein maybe tested and optimized using routine experimentation and the assays forselecting LT-beta-R blocking agents described herein.

[0068] Using the LT-beta-R ligand binding domain sequences within theextracellular domain shown in FIG. 1, amino acid sequence variants mayalso be constructed to modify the affinity of the soluble LT-betareceptor or fusion protein for LT ligand. The soluble LT-beta-Rmolecules of this invention can compete for surface LT ligand bindingwith endogenous cell surface LT-beta receptors. It is envisioned thatany soluble molecule comprising a LT-beta-R ligand binding domain thatcan compete with cell surface LT-beta receptors for LT ligand binding isa LT-beta-R blocking agent that falls within the scope of the presentinvention.

[0069] In another embodiment of this invention, antibodies directedagainst the human LT-beta receptor (anti-LT-beta-R Abs) function asLT-beta-R blocking agents for use in treating conditions that placeindividuals, including human, in, or at risk of, viral-induced systemicshock and respiratory distress. The anti-LT-beta-R Abs of this inventioncan be polyclonal or monoclonal (mAbs) and can be modified to optimizetheir ability to block LT-beta-R signalling, their in vivobioavailability, stability, or other desired traits.

[0070] Polyclonal antibody sera directed against the human LT-betareceptor are prepared using conventional techniques by injecting animalssuch as goats, rabbits, rats, hamsters or mice subcutaneously with ahuman LT-beta receptor-Fc fusion protein (Example 1 of U.S. Pat. No.5,925,351) in complete Freund's adjuvant, followed by boosterintraperitoneal or subcutaneous injection in incomplete Freund's.Polyclonal antisera containing the desired antibodies directed againstthe LT-beta receptor are screened by conventional immunologicalprocedures.

[0071] Mouse monoclonal antibodies (mAbs) directed against a humanLT-beta receptor-Fc fusion protein are prepared as described in U.S.Pat. No. 5,925,351, Example 5. A hybridoma cell line (BD.A8.AB9) whichproduces the mouse anti-human LT-beta-R mAb BDA8 was deposited on Jan.12, 1995 with the American Type Culture Collection (ATCC) (10801University Boulevard, Manassas, Va. 20110-2209) according to theprovisions of the Budapest Treaty, and was assigned the ATCC accessionnumber HB11798.

[0072] Various forms of anti-LT-beta-R antibodies can also be made usingstandard recombinant DNA techniques (Winter and Milstein, Nature, 349,pp. 293-99 (1991)). For example, “chimeric” antibodies can beconstructed in which the antigen binding domain from an animal antibodyis linked to a human constant domain (e.g. Cabilly et al., U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81, pp.6851-55 (1984)). Chimeric antibodies reduce the observed immunogenicresponses elicited by animal antibodies when used in human clinicaltreatments. In addition, recombinant “humanized antibodies” whichrecognize the LT-beta-R can be synthesized. Humanized antibodies arechimeras comprising mostly human IgG sequences into which the regionsresponsible for specific antigen-binding have been inserted (e.g. WO94/04679). Animals are immunized with the desired antigen, thecorresponding antibodies are isolated, and the portion of the variableregion sequences responsible for specific antigen binding are removed.The animal-derived antigen binding regions are then cloned into theappropriate position of human antibody genes in which the antigenbinding regions have been deleted. Humanized antibodies minimize the useof heterologous (inter-species) sequences in human antibodies, and areless likely to elicit immune responses in the treated subject.

[0073] Construction of different classes of recombinant anti-LT-beta-Rantibodies can also be accomplished by making chimeric or humanizedantibodies comprising the anti-LT-beta-R variable domains and humanconstant domains (CH1, CH2, CH3) isolated from different classes ofimmunoglobulins. For example, anti-LT-beta-R IgM antibodies withincreased antigen binding site valencies can be recombinantly producedby cloning the antigen binding site into vectors carrying the human muchain constant regions (Arulanandam et al., J. Exp. Med., 177, pp.1439-50 (1993); Lane et al., Eur. J. Immunol., 22, pp. 2573-78 (1993);Traunecker et al., Nature, 339, pp. 68-70 (1989)). In addition, standardrecombinant DNA techniques can be used to alter the binding affinitiesof recombinant antibodies with their antigens by altering amino acidresidues in the vicinity of the antigen binding sites. The antigenbinding affinity of a humanized antibody can be increased by mutagenesisbased on molecular modeling (Queen et al., Proc. Natl. Acad. Sci.U.S.A., 86, pp. 10029-33 (1989); WO 94/04679).

[0074] It may be desirable to increase or to decrease the affinity ofanti-LT-beta-R Abs for the LT-beta-R depending on the targeted tissuetype or the particular treatment schedule envisioned. For example, itmay be advantageous to treat a patient with constant levels ofanti-LT-beta-R Abs with reduced ability to signal through the LT-betapathway for semi-prophylactic treatments. Likewise, inhibitoryanti-LT-beta-R Abs with increased affinity for the LT-beta-R may beadvantageous for short-term treatments.

[0075] By testing other antibodies directed against the human LT-betareceptor, it is expected that additional anti-LT-beta-R antibodies thatfunction as LT-beta-R blocking agents in humans can be identified fortreating conditions that place individuals, including human, in, or atrisk of, viral-induced systemic shock and respiratory distress usingroutine experimentation and the assays described herein.

[0076] Another preferred embodiment of this invention involvescompositions and methods which comprise antibodies directed against LTligand that function as LT-beta-R blocking agents. As described abovefor the anti-LT-beta-R Abs, anti-LT ligand antibodies that function asLT-beta-R blocking agents can be polyclonal or monoclonal, and can bemodified according to routine procedures to modulate their antigenbinding properties and their immunogenicity. The anti-LT antibodies ofthis invention can be raised against either one of the two LT subunitsindividually, including soluble, mutant, altered and chimeric forms ofthe LT subunit. If LT subunits are used as the antigen, preferably theyare LT-beta subunits. If LT-alpha subunits are used, it is preferredthat the resulting anti-LT-alpha antibodies bind to surface LT ligandand do not cross-react with secreted LT-alpha or modulate TNF-R activity(according to the assays described in Example 3 of US Pat. No. 5,925,351).

[0077] Alternatively, antibodies directed against a homomeric (LT-beta)or a heteromeric (LT-alpha/62 ) complex comprising one or more LTsubunits can be raised and screened for activity as LT-beta-R blockingagents. Preferably, LT-alpha 1/beta 2 complexes are used as the antigen.As discussed above, it is preferred that the resulting anti-LT-alpha1/beta 2 antibodies bind to surfaceLT ligand without binding to secretedLT-alpha and without affecting TNF-R activity.

[0078] The production of polyclonal anti-human LT-alpha antibodies isdescribed in applicants' co-pending application (WO 94/13808).Monoclonal anti-LT-alpha and anti-LT-beta antibodies have also beendescribed (Browning et al., J. Immunol., 54, pp. 33-46 (1995)). Mouseanti-human LT-beta mAbs were prepared as described in Example 6 of U.S.Pat. No. 5,925,351. Hybridoma cell line (B9.C9.1) which produces themouse anti-human LT-beta-R mAb B9 was deposited on Jul. 21, 1995 withthe American Type Culture Collection (ATCC) (10801 University Boulevard,Manassas, Va. 20110-2209) according to the provisions of the BudapestTreaty, and was assigned the ATCC accession number 11962.

[0079] Monoclonal hamster anti-mouse LT-alpha/62 antibodies wereprepared as described in Example 7 of U.S. Pat. No. 5,925,351. Ahybridoma cell line (BB.F6.1) which produces the hamster anti-mouseLT-alpha/62 mAb BB.F6 was deposited on Jul. 21, 1995 with the AmericanType Culture Collection (ATCC) (10801 University Boulevard, Manassas,Va. 20110-2209) according to the provisions of the Budapest Treaty, andwas assigned the ATCC accession number MB11963.

[0080] A fluorescence-activated cell sorting (FACS) assay was developedto screen for antibodies directed against LT subunits and LT complexesthat can act as LT-beta-R blocking agents as described in Examples 6 and7 of U.S. Pat. No. 5,925,351. In this assay, soluble human LT-beta-R-Fcfusion protein is added to PMA-activated II-23 cells-which expresssurface LT complexes (Browning et al., J. Immunol., 154, pp. 33-46(1995))-in the presence of increasing amounts of the test antibody. Anantibody that can inhibit LT-beta receptor-ligand interaction by atleast 20% is selected as a LT-beta-R blocking agent.

[0081] Using a LT-alpha/beta complex rather than a LT subunit as anantigen to immunize an animal may lead to more efficient immunization,or may result in antibodies having higher affinities for surface LTligand. It is conceivable that by immunizing with the LT-alpha/62complex, antibodies which recognize amino acid residues on both theLT-alpha and the LT-beta subunits (e.g., residues that form anLT-alpha/62 cleft) can be isolated. By testing antibodies directedagainst human LT-alpha/62 heteromeric complexes, it is expected thatadditional anti-LT antibodies that function as LT-beta-R blocking agentsin humans can be identified using routine experimentation and the assaysdescribed herein.

[0082] Administration

[0083] The compositions described herein will be administered at aneffective dose in methods for treating viral-induced systemic shock andrespiratory distress in an individual. Determination of a preferredpharmaceutical formulation and a therapeutically efficient dose regimentfor a given application is well within the skill of the art takingintoconsideration, for example, the condition and weight of the patient,the extent of desired treatment and the tolerance of the patient for thetreatment. Doses of about 1 mg/kg of a soluble LT-beta-R are expected tobe suitable starting points for optimizing treatment doses.

[0084] Determination of a therapeutically effective dose can also beassessed by performing in vitro experiments that measure theconcentration of the LT-beta-R blocking agent required to coat targetcells (LT-beta-R or LT ligand-positive cells depending on the blockingagent) for 1 to 14 days. The receptor-ligand binding assays describedherein can be used to monitor the cell coating reaction. LT-beta-R or LTligand-positive cells can be separated from activated lymphocytepopulations using FACS. Based on the results of these in vitro bindingassays, a range of suitable LT-beta-R blocking agent concentrations canbe selected to test in animals according to the assays described herein.

[0085] Administration of the soluble LT-beta-R molecules, anti-LT ligandand anti-LT-beta-R Abs of this invention, alone or in combination,including isolated and purified forms of the antibodies or complexes,their salts or pharmaceutically acceptable derivatives thereof, may beaccomplished using any of the conventionally accepted modes ofadministration of agents which exhibit immunosuppressive activity.

[0086] The pharmaceutical compositions used in these therapies may alsobe in a variety of forms. These include, for example, solid, semi-solidand liquid dosage forms such as tablets, pills, powders, liquidsolutions or suspensions, suppositories, and injectable and infusiblesolutions. The preferred form depends on the intended mode ofadministration and therapeutic application.

[0087] Modes of administration may include oral, parenteral,subcutaneous, intravenous, intralesional or topical administration. Thesoluble LT-beta-R molecules, anti-LT ligand and anti-LT-beta-R Abs ofthis invention may, for example, be placed into sterile, isotonicformulations with or without cofactors which stimulate uptake orstability. The formulation is preferably liquid, or may be lyophilizedpowder. For example, the soluble LT-beta-R molecules, anti-LT ligand andanti-LT-beta-R Abs of this invention may be diluted with a formulationbuffer comprising 5.0 mg/ml citric acid monohydrate, 2.7 mg/ml trisodiumcitrate, 41 mg/ml mannitol, 1 mg/ml glycine and 1 mg/ml polysorbate 20.This solution can be lyophilized, stored under refrigeration andreconstituted prior to administration with sterile Water-For-Injection(USP).

[0088] The compositions also will preferably include conventionalpharmaceutically acceptable carriers well known in the art (see forexample Remington's Pharmaceutical Sciences, 16th Edition, 1980, MacPublishing Company). Such pharmaceutically acceptable carriers mayinclude other medicinal agents, carriers, genetic carriers, adjuvants,excipients, etc., such as human serum albumin or plasma preparations.The compositions are preferably in the form of a unit dose and willusually be administered one or more times a day.

[0089] The pharmaceutical compositions of this invention may also beadministered using microspheres, liposomes, other microparticulatedelivery systems or sustained release formulations placed in, near, orotherwise in communication with affected tissues or the bloodstream.Suitable examples of sustained releasecarriers include semipermeablepolymer matrices in the form of shaped articles such as suppositories ormicrocapsules. Implantable or microcapsular sustained release matricesinclude polylactides (U.S. Pat. No. 3,773,319; EP 58,481), copolymers ofL-glutamic acid and ethyl-L-glutamate (Sidman et al., Biopolymers, 22,pp. 547-56 (1985)); poly(2-hydroxyethyl-methacrylate) or ethylene vinylacetate (Langer et al., J. Biomed. Mater. Res., 15, pp. 167-277 (1981);Langer, Chem. Tech., 12, pp. 98-105 (1982)).

[0090] Liposomes containing soluble LT-beta-R molecules, anti-LT ligandand anti-LT-beta-R Abs of this invention, alone or in combination, canbe prepared by well-known methods (See, e.g. DE 3,218,121; Epstein etal., Proc. Natl. Acad. Sci. U.S.A., 82, pp. 3688-92 (1985); Hwang etal., Proc. Natl. Acad. Sci. U.S.A., 77, pp. 4030-34 (1980); U.S. Pat.Nos. 4,485,045 and 4,544,545). Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol. The proportion of cholesterolis selected to control the optimal rate of soluble LT-beta-R molecule,anti-LT ligand and anti-LT-beta-R Ab release.

[0091] The soluble LT-beta-R molecules, anti-LT ligand andanti-LT-beta-R Abs of this invention may also be attached to liposomescontaining other LT-beta-R blocking agents, immunosuppressive agents orcytokines to modulate the LT-beta-R blocking activity. Attachment ofLT-beta-R molecules, anti-LT ligand and anti-LT-beta-R Abs to liposomesmay be accomplished by any known cross-linking agent such asheterobifunctional cross-linking agents that have been widely used tocouple toxins or chemotherapeutic agents to antibodies for targeteddelivery.conjugation to liposomes can also be accomplished using thecarbohydrate-directed cross-linking reagent 4-(4-maleimidophenyl)butyric acid hydrazide (MPBH) (Duzgunes et al., J. Cell. Biochem. Abst.Suppl. 16E 77 (1992)).

[0092] The LT-beta-R blocking agents of the compositions and methods ofthis invention can be modified to obtain a desirable level of LT-beta-Rsignalling depending on the condition, disorder or disease beingtreated. It is envisioned that the absolute level of LT-beta-Rsignalling can be fine-tuned by manipulating the concentration and theaffinities of the LT-beta-R blocking agents for their respectivemolecular targets. For example, in one embodiment of this invention,compositions comprising soluble LT-beta-R molecules are administered toa subject. The soluble LT-beta receptor can effectively compete withcell surface LT-beta receptors for binding surface LT ligands. Theability to compete with surface LT ligands depends on the relativeconcentrations of the soluble and the cell surface LT-beta-R molecules,and on their relative affinities for ligand binding.

[0093] Soluble LT-beta-R molecules harboring mutations that increase ordecrease the binding affinity of that mutant soluble LT-beta-R withsurface LT ligand can be made using standard recombinant DNA techniqueswell known to those of skill in the art. Large numbers of molecules withsite-directed or random mutations can be tested for their ability to actas LT-beta-R blocking agents using routine experimentation and thetechniques described herein. Similarly, in another embodiment of thisinvention, antibodies directed against either the LT-beta receptor orone or more of the LT ligand subunits function as LT-beta-R blockingagents. The ability for these antibodies to block LT-beta receptorsignalling can be modified by mutation, chemical modification or byother methods that can vary the effective concentration or activity ofthe antibody delivered to the subject.

[0094] Uses

[0095] As a general matter, the methods of the present invention may beutilized for inducing an antiviral response in an individual comprisingadministering to the individual an effective amount of a LT-B blockingagent and a pharmaceutically acceptable carrier. The viral response tobe treated may be caused by any number of known viruses, including butnot limited to Sin Nombre (SNV), Ebola, Marburg, Lassa, and Dengue.

Equivalents

[0096] The invention may be embodied in other specific forms withoutdeparting from the spirit or essential characteristics thereof. Theforegoing embodiments are therefore to be considered in all respectsillustrative of, rather than limiting on, the invention disclosedherein. Scope of the invention thus is indicated by the appended claimsrather than by the foregoing description, and all changes which comewithin the meaning and range of equivalency of the claims are intendedto be embraced therein.

EXAMPLE

[0097] Tumor necrosis factor (TNFα) plays a key role in facilitatingacute shock responses to viral infections and other immunogens (K. C. F.Sheehan, N. H. Ruddle, and R. D. Schreiber., J. Immunol., 142, 3884(1989); G. W. H. Wong and D. V. Goeddel Nature 323, 819 (1986); B.Beutler, I. W. Milsark, A. Cerami, Science 229, 869 (1985); F. Mackay,P. R. Bourdon, D. A. Griffiths, et al. J. Immunol. 159, 3299 (1997); P.D. Crowe, T. L. VanArsdale, B. N. Walter, et al. Science 264, 707(1994)). During episodes of Dengue Fever involving shock, levels of TNFαin sera from patients are elevated as are levels of soluble TNFR-75 (D.Hober, et al., J. Trop. Med. Hyg., 48, 324 (1993); D. B. Bethell, K.Flobbe, C. X. T. Phuong, et al., J. Infect. Dis., 177, 778 (1998)). Wemeasured TNFα levels in the sera of mice infected with a variant oflymphocytic choriomeningitis virus, LCMV, Clone 13 (LCMV-13) (HH, II).TNFα levels in the sera of mice infected with LCMV-13 were found to bejust above the level of detection for the assay until day 4 postinfection (Serum TNFα levels were measured by ELISA assay (GenzymeCorporation, catalog number 80-2802-00)). On days 5 and 6, when thedisease is at its peak, soluble TNFα levels in the serum increased 3-6fold above normal (data not shown). We therefore chose to block TNFαfunction by using a monoclonal antibody, TN3-19.12, which is known tobind both secreted TNFα, thus causing its depletion from the mouse asverified by ELISA (K. C. F. Sheehan, N. H. Ruddle, and R. D. Schreiber.,J. Immunol., 142, 3884 (1989) G. W. H. Wong and D. V. Goeddel Nature323, 819 (1986); B. Beutler, I. W. Milsark, A. Cerami, Science 229, 869(1985); F. Mackay, P. R. Bourdon, D. A. Griffiths, et al. J. Immunol.159, 3299 (1997); P. D. Crowe, T. L. VanArsdale, B. N. Walter, et al.Science 264, 707 (1994); D. Hober, et al., J. Trop. Med. Hyg., 48, 324(1993); D. B. Bethell, K. Flobbe, C. X. T. Phuong, et al., J. Infect.Dis., 177, 778 (1998)). Serum TNFα levels were measured by ELISA assay(Genzyme Corporation, catalog number 80-2802-00). NZB mice were given2.5×10⁶ pfu Cl 13 i.v. followed by two i.p. injections containing 250 μgof TN3-19.12 antibody in endotoxin free PBS (see reference S) on days 1and day 4 post-infection. Control mice were injected with the samevolume of PBS lacking antibody on the same days. This treatment(anti-TNF) had little effect on the survival rate of these mice (FIG.3). Lymphotoxin alpha (LTα), also known as TNFβ, though it sharesidentical receptors and many of its biological effects with TNFα, is notrecognized by this antibody (F. Mackay, P. R. Bourdon, D. A. Griffiths,et al. J. Immunol. 159, 3299 (1997). It is possible that targeting bothTNFα and LTα are required to increase survival rates. To test thishypothesis, we used the above TN3-19.12 mAb and a receptor fusionprotein that fused the extracellular domain of the TNF p55 receptor toCH2 and CH3 domains of human IgG1 (TNFR55-Ig)(W. R. Force, B. N. Walter,C. Hession, et. al., J. Immunol., 155, 5280 (1995); G. T. Miller, P. S.Hochman, W. Meier, et. al., JEM., 178, 211 (1993); J. L. Browning, I.Dougas, A. Ngam-ek, et al., J. Immunol., 154:33 (1995). Mice weretreated as described in reference R. For the triple treated group,TNFR55-Ig and LTβR-Ig proteins were given on day 0 and day 3post-infection, i.p., in 200 μg amounts. Control mice were given humanantibody used in the synthesis of these fusion proteins (AY1943-29) onthe same days in identical amounts. Mice receiving LTβR-Ig only weretreated identically, except the TNFR55-Ig injections were omitted). Thistreatment also did not significantly alter survival rates in LCMV-13infected NZB mice (See anti-TNF and TNFR55-Ig group). The membrane formof lymphotoxin, a heteromer of LTα and LTβ, does not recognize TNFR-75or TNFR-55 but rather binds to a third receptor called LTβR (15). Weelected to use a fusion protein containing the LTβR extracellular domainalso attached to CH2 and CH3 domains of human IgG1 (LTβR-Ig). Treatmentof the mice with anti-TNFα mAb, TNFR55-Ig and LTβR-Ig (triple treatmentor TNFR55-IG and LTβR-Ig) resulted in a dramatic increase in survival,to 80% and 70% respectively. In contrast, only 20% of mice treated withanti-TNFα mAb and TNFR55-Ig survived infection. Recently a second ligandfor LTβR, LIGHT, was identified (D. N. Mauri, R. Ebner, R. I.Montgomery, et al. Immunity 8, 21 (1998); R. I. Montgomery, M. S.Warner, B. Lum, et al. Cell 87, 427 (1996)). LIGHT has also been shownto bind the herpesvirus entry mediator (HVEM), a type I transmembraneprotein with significant homology to members of the TNFR family that isexpressed on activated CD4 and CD8 T cells (D. N. Mauri, R. Ebner, R. I.Montgomery, et al. Immunity 8, 21 (1998); R. I. Montgomery, M. S.Warner, B. Lum, et al. Cell 87, 427 (1996)). Based on results presentedhere, prevention of LTβR signaling and potentially HVEM signaling by thebinding of LTβ₂α₁ and LIGHT by LTβR-Ig was likely responsible for mostof the effect seen in the triple treatment group. We affirmed thishypothesis by treating LCMV-13 infected NZB mice with just the LTβR-Igfusion protein. The survival rate of mice in this group (73%) was almostas high as the triple treated group (FIG. 3). Taken together, these datarepresent the first demonstration that the LTβR and/or HVEM signalingpathway is involved in the orchestration of an acute lethal diseaseinvolving systemic shock and respiratory distress.

[0098] In an effort to determine the mechanism of survival behind LTβblockage treatment, both CD8/tetramer co-staining for NP118 specific Tcells, the dominant CD8 epitope in the NZB L^(D) system, andintracellular staining for interferon gamma production by spleenocytesstimulated with NP118 peptide were performed on samples from LCMV-13infected NZB mice who were treated with control antibody, LTβR-Ig alone,or triple treated. FIG. 4 demonstrates a reduction in the number ofNP118 specific CD8 T cells with the greatest effect seen in the tripletreatment mice. In mice treated with control antibody, only 10% oftetramer positive cells actively produced INF_(γ). The emergence ofanergic T cells during LCMV-13 infection has been previously documentedand is likely due to high levels of viral antigen in the mouse (FIG. 1).Not only has the number of NP118 specific cells declined in the LTβR-Igtreated mice, but the percentage of those cells producing INF_(γ)wasalso reduced. This effect was even more pronounced in the tripletreatment group. Thus it is possible that the CD8 compartment may be thesource of this lethal NZB response to LCMV-13 infection. The fact thatactivated CD8s are known to display LTβ₂α₁ is consistent with thishypothesis (Y. Abe, A. Horiuchi, Y. Osuka, et al., Lymph. Ctyok. Res.,11, 115 (1992); C. F. Ware, P. D. Crowe, M. H. Grayson, et al., J.Immunol., 149, 3881 (1992); J. L. Browning, A. Ngam-ek, P. Lawton, etal., Cell, 72, 847 (1993)). To support this assertion, we depletedinfected NZB mice of their CD8 or CD4 positive T cells in vivo (Male NZBmice were given 2.5×10⁶ pfu LCMV-13 i.v. followed by two 500 μl i.p.injections of anti T cell antibody. The mAb Lyt2.43 was used to depleteCD8⁺T cells while the GK1.5 (Ml) antibody was used for CD4⁺T celldepletion. Both antibodies were prepared by an ammonium sulfateprecipitation from hybridoma supernatants followed by dialysis againstPBS. FACS analysis was used to verify the depletion in several of themice.). Depletion of CD4 T cells did not increase survival. In contrast,depletion of CD8 T cells resulted in 100% survival in the absence ofdisease symptoms unlike the LTβR-Ig treated mice (FIG. 5). Because viraltiters in several tissues of CD8 depleted mice were higher than thosenot treated, it is likely that death resulted from a toxic immuneresponse mediated by CD8 T cells rather than from destruction of tissuesby viral infection.

[0099] We have reported here that NZB mice when infected with a highdose of LCMV-13 intravenously develop an acute, rapidly progressivedisease that shares several common traits with Ebola, Marburg, Lassa,Dengue, and Sin Nombre infections. Lethality of this illness wasdependent on the presence of CD8⁺T cells which are known to expressTNFα, LTα, and LTβ when activated. Though this is an encouragingfinding, treatment of viral infection by depletion of CD8⁺T cells wouldnot be advisable. Such treatment could leave patients vulnerable toother opportunistic infections. Furthermore, since viral clearance isunlikely in the absence of CTLs the risk of the patient tolerizing tothe virus upon re-establishment of the CD8⁺compartment is very real. Wehave shown that blockage of the LTβR/HVEM pathways by administration ofLTβR-Ig represents a powerful treatment that is transient in nature,with rapid recovery to homeostasis once treatment is stopped (Mackay andBrowning, unpublished). Surviving mice treated in this manner eventuallycleared virus from tissues tested (data not shown) and no longer showsigns of disease.

[0100] These data represent the first demonstration that LTβR signalingplays an important role in antiviral responses and CD8 T cell function.The lymphotoxin system is intimately linked to organization of lymphoidarchitecture most likely via control of the expression of severalchemokines that direct T and B cell organization (. Chaplin et al. Curr.Opin. Immunol. 10, 289 (1998), J. Cyster, in press). The maturefunctional status of follicular dendritic cells is maintained byconstant B cell signaling and these cells disappear within one day uponcessation of the LTβR signaling. These cells are critical for thepresentation of antigen to the B and T cell compartments. A reasonablespeculation is that some aspect of antigen presentation to CD8 cells orthe proper positioning of these cells in a chemokine gradient duringmaturation is prevented by disruption of LTβR signaling. Previousstudies of LT function have focused primarily on B cell biology and theinvolvement in a T cell function was unforeseen. Either LT hasadditional functions or these data reflect a role for the novel ligandLIGHT. What role HVEM and LIGHT may play in the progression of thedisease documented here is unclear at present.

What is claimed is:
 1. A method of inducing an antiviral response in anindividual comprising administering to the individual an effectiveamount of an agent which blocks the binding of lymphotoxin-β to itsreceptor, and a pharmaceutically acceptable carrier.
 2. A method ofclaim 1 wherein said agent is a LT-beta-R blocking agent.
 3. The methodof claim 2 wherein said agent is an antibody against the lymphotoxin-βreceptor or a soluble lymphotoxin-β receptor.
 4. The method of claim 3wherein said agent is a lymphotoxin-β receptor/Ig fusion protein.
 5. Themethod of claim 1 wherein said agent is a soluble lymphotoxin-β or anantibody against lymphotoxin-β.
 6. A method of inducing an antiviralresponse in an individual comprising administering to the individual aneffective amount of an agent which blocks the lymphotoxin-β-receptorand/or HVEM signaling pathway.
 7. The method of claims 1-6 wherein saidindividual is infected with Sin Nombre Virus, Ebola virus, Marburgvirus, Lassa virus or Dengue.
 8. The method of claim 7 wherein the agentis a lymphotoxin-β-R/Ig fusion protein.